AU2017328310B2

AU2017328310B2 - Antibody specifically binding to IL-17A and functional fragment thereof

Info

Publication number: AU2017328310B2
Application number: AU2017328310A
Authority: AU
Inventors: Mengxie JIN; Jiawang Liu; Nanmeng SONG; Dongge YANG; Yang Yang; Yaping Yang; Hongjuan ZHANG
Original assignee: Beijing Hanmi Pharmaceutical Co Ltd
Current assignee: Beijing Hanmi Pharmaceutical Co Ltd
Priority date: 2016-09-14
Filing date: 2017-09-08
Publication date: 2020-10-15
Anticipated expiration: 2037-09-08
Also published as: WO2018050028A1; KR20190045348A; JP2020500206A; IL265350B1; CA3036913A1; AU2017328310A1; SA519401307B1; JP6802381B2; PE20191075A1; CN107556382B; MY185578A; ECSP19018356A; NZ752189A; US20190256588A1; BR112019004990A2; CN109715660A; KR102329490B1; US11072651B2; CL2019000659A1; AU2017328310A9

Abstract

An antibody specifically binding to IL-17A and a functional fragment thereof. The antibody or functional fragment thereof includes an IL-17A chimeric antibody and a functional fragment thereof, and an IL-17A humanized antibody and a functional fragment thereof.

Description

ANTIBODY SPECIFICALLY BINDING TO IL-17A AND FUNCTIONAL FRAGMENT

THEREOF

[0001] The present application claims priority to Chinese Patent Application No.

CN201610827097.2, filed on September 14, 2016 with State Intellectual Property Office and

entitled "ANTIBODY SPECIFICALLY BINDING TO IL-17A AND FUNCTIONAL

FRAGMENT THEREOF", the entire content of which is incorporated herein by reference.

FIELD

[0002] The present disclosure relates to the field of medical biotechnology and humanized

antibody engineering research, and in particular to an antibody specifically binding to IL-17A

and functional fragments thereof.

BACKGROUND

[0003] Any discussion of the prior art throughout the specification should in no way be

considered as an admission that such prior art is widely known or forms part of common general

knowledge in the field.

[0003A]Interleukin-17A (IL-17 or IL-I7A) is a pro-inflammatory cytokine produced primarily

by Th17 cells and is the most representative member of the IL-17 family (Miossec P, Kolls JK.

Nat. Rev. Drug. Discov., 2012,11: 763-776). After binding to IL-17A receptor (IL-17RA),

IL-17A may induce the expression of inflammatory cytokines and chemokines in a variety of

cells (such as fibroblasts, epithelial cells, and endothelial cells), playing an important role in

body immunological defense (Lin Y, Ritchea S, Logar A. Immunity, 2009, 31:799-810).

[0004] However, over-expressed IL-17A may cause many inflammatory diseases. For example,

IL-17A has effects on macrophages and DC cells, inducing high expression of IL-i, IL-6, TNF

and CRP, resulting in inflammatory reaction, and involving in the pathological processes of psoriasis and transplant rejection. IL-17A has effects on endothelial cells, inducing high expression of IL-6, MMP and coagulation factors, resulting in vascular activation, and involving in pathological processes of reperfusion injury, thrombosis and atherosclerosis. IL-17A has effects on fibroblasts, inducing high expression of IL-6, chemokines, growth factors and MMP, resulting in matrix destruction, and involving in the pathological process of multiple sclerosis and Crohn's disease. IL-17A has effects on osteoblasts and chondrocytes, inducing RANKL,

MMP and osteoclast genesis, resulting in bone erosion and cartilage damage, and involving in

the pathological process of rheumatoid arthritis and periodontal disease (N Engl J Med. 2009

Aug 27; 361(9):888-98. Nat Rev Drug Discov. 2012 Oct; 11(10):763-76. Semin Arthritis Rheum.

2013 Oct; 43(2):158-70. Trends Mol Med. 2016 Mar; 22(3):230-41).

[0005] IL-17A neutralizing antibody can inhibit the high expression of IL-17A in synovial

tissue of patients with rheumatoid arthritis and reduce the production of important inflammatory

factor IL-6 (Chabaud M, Durand JM, Buchs N, et al. Arthritis Rheum. 1999,42: 963-70). It has

been demonstrated by many animal model experiments of autoimmune diseases that the use of

antibodies to neutralize IL-17A can effectively inhibit the pathological development of

inflammation (Lubberts E, Koenders MI, Oppers-Walgreen B, et al. Arthritis Rheum., 2004, 50:

650-659).

[0006] Currently, IL-17A-related antibody drugs, Secukinumab (IL-17A targeting antibody)

and Ixekizumab (IL-17A targeting antibody), have been approved for marketing, which are both

used for the treatment of psoriasis. However, results of drug clinical trial have shown that these

drugs do not show the expected therapeutic effect for certain chronic inflammatory diseases such

as rheumatoid arthritis (Kellner H,Ther Adv Musculoskelet Dis., 2013, 5: 141-152).

[0007] In view of this, the present disclosure has been specifically proposed.

SUMMARY

[0008] Unless the context clearly requires otherwise, throughout the description and the claims,

2 (followed by page 2a) the words 'comprise', 'comprising' and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say in the sense of "including but not limited to".

[0008A]The present disclosure is based on an obtained parental anti-human IL-17A murine monoclonal antibody having the ability to specifically bind to human IL-I7A protein, by cloning, identification and gene structure analysis to determine its CDR region, construct corresponding chimeric antibody and humanized antibody, establish corresponding eukaryotic cell expression

[FOLLOWED BY PAGE 3]

2a

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system and produce and purify the chimeric antibody and the humanized antibody.

[0009] In order to achieve the above-mentioned goal of the present disclosure, the following

technical solutions are specially adopted:

[0010] An antibody capable of specifically binding to IL-17A and a functional fragment thereof,

wherein the antibody or the functional fragment thereof comprise a light chain and a heavy

chain;

[0011] the light chain comprises a light chain CDR consisting of CDR-L1, CDR-L2 and

CDR-L3; the heavy chain comprises a heavy chain CDR consisting of CDR-H1, CDR-H2 and

CDR-H3;

[0012] the amino acid sequences of the CDR-L1, CDR-L2 and CDR-L3 are respectively set

forth in SEQ ID NO: 1, 2 and 3; the amino acid sequences of the CDR-H1, CDR-H2 and

CDR-H3 are respectively set forth in SEQ ID NO: 4, 5 and 6.

[0013] Preferably, the antibody or the functional fragment thereof includes an IL-17A chimeric

antibody and a functional fragment thereof, and an IL-17A humanized antibody and a functional

fragment thereof.

[0014] Preferably, the antibody or the functional fragment thereof includes an IL-17A chimeric

fragment thereof.

[0015] That is, the antibody or the functional fragment thereof includes an IL-17A chimeric

antibody and a functional fragment thereof, or the antibody or the functional fragment thereof

includes an IL-17A humanized antibody and a functional fragment thereof.

[0016] It is well known in the art that both the binding specificity and affinity of an antibody

are mainly determined by the CDR, and the amino acid sequence of the non-CDR region can be

easily changed according to the well-known existing techniques to obtain a variant having

similar biological activities. In the present disclosure, the monoclonal antibody variants have

CDR sequences identical to the CDR sequences of above-mentioned humanized antibodies, thus,

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they have similar biological activities.

[0017] Preferably, the antibody and the functional fragment thereof as described above,

wherein the antibody comprises a constant region sequence of any one selected from the group

consisting of human IgGI, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0018] Preferably, the antibody and the functional fragment thereof as described above,

wherein the functional fragment comprises one or more selected from the group consisting of

F(ab') 2 , Fab', Fab, Fv, scFv, bispecific antibody and antibody minimal recognition unit.

[0019] The "functional fragment" of the present disclosure specifically refers to an antibody

fragment having the same specificity to IL-17A as that of the parent antibody. In addition to the

above mentioned functional fragments, any fragment of which half-life has been increased may

be also included.

[0020] scFv (sc = single strand), bispecific antibody (diabodies).

[0021] These functional fragments typically have the same binding specificity as the antibody

from which they are derived. One ordinary skill in the art can learn from what is described in the

specification of the present disclosure that the antibody fragment of the present disclosure and

obtain the above mentioned function fragment by a method such as enzymatic digestion

(including pepsin or papain) and/or a method of chemically reducing split disulfide bonds.

[0022] The antibody fragments can also be obtained by peptide synthesis by recombinant

genetic techniques, which are also known to those having ordinary skill in the art, or by

automated peptide synthesizers such as an automated peptide synthesizer sold by such as

Applied BioSystems.

[0023] Preferably, the antibody and the functional fragment thereof as described above,

wherein the amino acid sequences of light chain variable region and heavy chain variable region

of the IL-17A chimeric antibody and the functional fragment thereof are respectively set forth in

SEQ ID NO: 7 and SEQ ID NO:8;

[0024] Further preferably, the antibody and the functional fragment thereof as described above,

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wherein the amino acid sequence of the light chain constant region and the heavy chain constant

region of the IL-I7A chimeric antibody and functional fragment thereof are respectively set forth

in SEQID NO: 9 and SEQID NO: 10.

[0025] Preferably, the antibody and the functional fragment thereof as described above,

wherein light chain framework region of the IL-17A humanized antibody and the functional

fragment thereof comprises FR-L, FR-L2, FR-L3 and FR-L4, and heavy chain framework

region of the IL-17A humanized antibody and the functional fragment thereof comprises FR-HI,

FR-H2, FR-H3 and FR-H4.

[0026] The amino acid sequences of FR-Li, FR-L2, FR-L3 and FR-L4 are set forth in SEQ ID

NO: I Ito 14 respectively.

[0027] The amino acid sequences of the FR-H, FR-H2, FR-H3 and FR-H4 are set forth in

SEQ ID NO: 15 to 18 respectively.

[0028] Usually, when transplanting CDRs of a murine antibody to a human framework,

selection of a human framework with high sequence homology will have a certain success rate.

However, studies have shown that many CDR grafts require a back mutation to restore certain

antibody activity. How to choose the right human source framework is the major bottleneck.

[0029] The CDR is the major relevant site for antigen binding, but in most cases, the FR

(framework region) has a significant influence on the conformation of the binding site. In order

to obtain a high affinity humanized antibody, in the present disclosure, a suitable FR region is

selected and the relevant FR residue is reversed back to the original murine amino acid or a

amino acid presented in human and having the same function.

[0030] Preferably, the FR-Li is selected from the amino acid sequence set forth in SEQ ID NO:

I Iand the amino sequence having the following substitution or a combination thereof:

[0031] the 1t amino acid D is replacedbyI;

[0032] the 2" amino acid V is replaced by I;

[0033] the FR-L2 is selected from the amino acid sequence set forth in SEQ ID NO: 12 and the

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amino sequence having the following substitution or a combination thereof:

[0034] the 4 th amino acid F is replaced by Y;

[0035] the 14th amino acid R is replaced by L;

[0036] the FR-L3 is selected from the amino acid sequence set forth in SEQ ID NO: 13 and the

amino sequence having the following substitution or a combination thereof:

[0037] the 3 5 th amino acid Y is replaced by F;

[0038] the FR-L4 is selected from the amino acid sequence set forth in SEQ ID NO: 14;

[0039] the FR-Hi is selected from the amino acid sequence set forth in SEQ ID NO: 15 and the

amino sequence having the following substitution or a combination thereof:

[0040] the 4 th amino acid L is replaced by V;

[0041] the FR-H2 is selected from the amino acid sequence set forth in SEQ ID NO: 16 and the

amino sequence having the following substitution or a combination thereof:

[0042] the 1 5 th amino acid I is replaced by M;

[0043] the FR-H3 is selected from the amino acid sequence set forth in SEQ ID NO: 17 and the

amino sequence having the following substitution or a combination thereof:

[0044] the 2"d amino acid V is replaced by I;

[0045] the 6 th amino acid V is replaced by R;

[0046] the FR-H4 is selected from the amino acid sequence set forth in SEQ ID NO: 18;

[0047] preferably, light chain variable region sequence of the IL-17A humanized antibody and

the functional fragment thereof is one selected from SEQ ID NO: 19-26;

[0048] preferably, heavy chain variable region sequence of the IL-17A humanized antibody and

the functional fragment thereof is one selected from SEQ ID NO: 27-34;

[0049] more preferably, the light chain variable region sequence of the IL-17A humanized

antibody and the functional fragment thereof is set forth in SEQ ID NO: 19; the corresponding

heavy chain variable region sequence is set forth in SEQ ID NO: 27;

[0050] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

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and the functional fragment thereof is set forth in SEQ ID NO: 20; the corresponding heavy

chain variable region sequence is set forth in SEQID NO: 28;

[0051] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

and the functional fragment thereof is set forth in SEQ ID NO: 21; the corresponding heavy

chain variable region sequence is set forth in SEQ ID NO: 29;

[0052] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

and the functional fragment thereof is set forth in SEQ ID NO: 22; the corresponding heavy

chain variable region sequence is set forth in SEQ ID NO: 30;

[0053] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

chain variable region sequence is set forth in SEQ ID NO: 31;

[0054] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

and the functional fragment thereof is set forth in SEQ ID NO: 23; the corresponding heavy

chain variable region sequence is set forth in SEQ ID NO: 32;

[0055] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

and the functional fragment thereof is set forth in SEQ ID NO: 24; the corresponding heavy

chain variable region sequence is set forth in SEQ ID NO: 33;

[0056] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

and the functional fragment thereof is set forth in SEQ ID NO: 25; the corresponding heavy

chain variable region sequence is set forth in SEQ ID NO: 32;

[0057] alternatively, the light chain variable region sequence of the IL-17A humanized antibody

and the functional fragment thereof is set forth in SEQ ID NO: 26; the corresponding heavy

chain variable region sequence is set forth in SEQ ID NO: 34;

[0058] more preferably, the amino acid sequences of the light chain constant region and the

heavy chain constant region of the IL-17A humanized antibody and the functional fragment

thereof are respectively set forth in SEQ ID NO: 9 and SEQ ID NO: 10.

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[0059] The light chain constant region of the IL-17A humanized antibody and the functional

fragment thereof is selected from the light chain constant region of human Kappa antibody, of

which the amino acid sequence is set forth in SEQID NO: 9.

[0060] The heavy chain constant region of the IL-17A humanized antibody and the functional

fragment thereof is selected from the heavy chain constant region of human IgGlantibody, of

which the amino acid sequence is set forth in SEQID NO: 10.

[0061] It should be noted that, in addition to the above-mentioned amino acid sequences in the

present application, the production of chimeric antibodies and humanized antibodies can be

achieved by any method known by those having ordinary skill in the art, such as by designing

recombinant humanized antibody based on sequenced CDRs of murine antibodies, the murine

antibody is secreted by myeloma cells from immunized mice or by myeloma cells fused to

splenocytes of other species which fused to myeloma cells. The immunized animal may include

a transgenic mouse having a human immunoglobulin locus which can directly produce a human

antibody. Another possible embodiment may include screening the library using phage display

technology.

[0062] An isolated nucleic acid molecule selected from:

[0063] A) DNA or RNA, encoding the antibody and the functional fragment thereof as

described above; and

[0064] B) a nucleic acid complementary to the nucleic acid as defined in A).

[0065] A vector, which contains a nucleic acid molecule as described above.

[0066] The present disclosure further provides at least one nuclear construct encoding a nucleic

acid molecule as described above, preferably a vector, more preferably an expression vector,

such as a plasmid, which is described in one embodiment of the present application.

[0067] A host cell, which is transformed with a vector as described above.

[0068] The host cell is a eukaryotic cell, such as a mammalian cell.

[0069] A method of producing an antibody capable of specifically binding to IL-17A and a

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functional fragment thereof includes the following steps:

[0070] culturing host cells as described above in a medium and under suitable culture

conditions; and

[0071] recovering produced antibody and its functional fragments from the culture

medium or from the cultured host cells.

[0072] A composition, comprising the antibody and/or the functional fragment thereof, or a

compound of the antibody and/or the functional fragment thereof together with other components,

as an active ingredient.

[0073] Preferably, the composition as described above, the antibody and the functional

fragment thereof are coupled to at least one diagnostic agent and/or therapeutic agent to form an

immunoconjugate.

[0074] Preferably, the diagnostic agent is one or more selected from the group consisting of a

radionuclide, a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent label, a

chemiluminescent label, an ultrasound contrast agent, and a photosensitizer.

[0075] Preferably, the radionuclide is one or more selected from the group consisting of ""In,

"In, "7Lu, 18F, 52Fe, 62Cu, 14Cu, 17Cu, 67Ga, 68Ga, 6Y, 9Y, 89Zr, 94mTc, 94Tc, 99mTc, 120 1,23 1,24 125 1,31 154-158Gd, 32P, C, "N, o, 18 Re, Re, 5Mn, 52mMn, 5Co, 72As, 7Br, 76Br, 82mRb

and 83Sr.

[0076] Preferably, the paramagnetic ion is one or more selected from the group consisting of

chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II),

neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III),

dysprosium (III), holmium (III) and erbium (III).

[0077] Preferably, the fluorescent label is one or more selected from the group consisting of

Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa555,Alexa 647, AMCA, aminoacridine,

BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein,

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5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine,

6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7,

6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole),

Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid,

terephthalic acid, isophthalic acid, cresol fast purple, cresyl violet, brilliant cresyl blue,

4-Aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl

fluorescein, rare earth metal cryptate, tri-bipyridyldiamine oxime, europium cryptate compound

or chelate, diamine, dicyanine, La Jolla blue dye, allophycocyanin, allococyanin B, phycocyanin

C, phycocyanin R, thiamine, R-phycoerythrin, C-Phycocyanin, phycoerythrin R, REG,

rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT

(tetramethylrhodamine isothiol), tetramethylrhodamine and Texas Red.

[0078] Preferably, the therapeutic agent is one or more selected from the group consisting of a

naked antibody, a cytotoxic agent, a drug, a radionuclide, a boron atom, an immunomodulator, an

anti-apoptotic agent, a photosensitizing therapeutic, an immunoconjugates and a oligonucleotide.

[0079] Preferably, the drug is one or more selected from the group consisting of methotrexate,

fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide,

thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D,

doxorubicin, daunorubicin, vinblastine, paclitaxel, cephalotaxus alkaloids and L-asparaginase.

[0080] Preferably, the oligonucleotide is one or more selected from the group consisting of

shRNA, miRNA and siRNA.

[0081] Preferably, the immunomodulator is one or more selected from the group consisting of a

cytokine, a chemokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a

colony stimulating factor (CSF), an interferon, an erythropoietin, a thrombopoietin, an

interleukin (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony

stimulating factor (GM-CSF) and stem cell growth factor.

[0082] Wherein, the cytokine is preferably one or more selected from the group consisting of

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human growth hormone, N-methionyl human growth hormone, bovine growth hormone,

parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle-stimulating

hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), liver growth

factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, tumor

necrosis factor-a, tumor necrosis factor-p, Mullerian inhibitor, mouse gonadotropin-related

peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO),

NGF-j, platelet-growth factor, TGF-a, TGF-j, insulin-like growth factor-I, insulin-like growth

factor-II, erythropoietin (EPO), osteoinductive factor, interferon-a, interferon-p, interferon-y,

macrophage-CSF (M-CSF), IL-, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,

IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin,

thrombospondin, endostatin, tumor necrosis factor and LT.

[0083] The chemokine is preferably one or more selected from the group consisting of

RANTES, MCAF, MIP1-a, MIP1-P, and IP-10.

[0084] Preferably, the radionuclide is one or more selected from the group consisting of I"In, At, 177Lu, 211Bi, 212Bi, 213Bi, 211At, 62Cu, 67Cu, 90 125 131 133 32P 33 47Sc, 11Ag, 67Ga,

153 161 152 166 161 166 186 18 189 211 212 223 225 77 8 Sm, Tb, Dy, Dy, Ho, Ho, Re, '"Re, Re, 2Pb, Pb, Ra, Ac, As, 89Sr,

99Mo, 105Rh, 149Pm, 169Er, 194Ir, 58Co, 80mBr, 99mTc, 103 mRh, 0 9Pt, 11 9 Sb, 189mOs, 192r, 219Rn,

215Po, 221Fr, 25sFm, "C, 1N, o, 7Br, 198Au, 199Au, 224Ac, 7Br, 13mIn, 95Ru, 97Ru, 103Ru, 105Ru,

17Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 142Pr, 143Pr, 16 1 Tb, 57 Co,

5C8o, 5Cr, 59Fe, 7Se, 201TI, 76Br and 169Yb.

[0085] Use of the composition as described above for the manufacture of a medicament in

prevention and/or treatment of a disease associated with overexpression and/or over-release of

IL-I7A.

[0086] Preferably, the disease is one or more selected from the group consisting of airway

inflammation, asthma, bronchial asthma, allergic asthma, chronic obstructive pulmonary disease,

idiopathic pulmonary fibrosis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis,

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psoriatic arthritis, psoriasis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus,

lupus nephritis, scleroderma, ulcerative colitis, inflammatory bowel disease, uveitis, helicobacter

pylori-associated gastritis, osteoporosis, bone erosion, intraperitoneal abscess and adhesions,

Addison's disease, gamma globulin deficiency, alopecia areata, celiac disease, Chagas disease,

Crohn's disease, allograft rejection, Behcet's disease, sepsis, septic or endotoxin shock and

ischemia.

[0087] Use of the antibody capable of specifically binding to IL-17A and the functional

fragment thereof as described above for the manufacture of a medicament in prevention and/or

treatment of a disease associated with overexpression and/or over-release of IL-17A.

[0088] A drug for prevention and/or treatment of a disease associated with overexpression

and/or over-release of IL-I7A, comprising the antibody capable of specifically binding to IL-I7A

and the functional fragment thereof, and a pharmaceutically acceptable carrier.

[0089] Alternatively, the drug comprises the composition as described above and a

pharmaceutically acceptable carrier.

[0090] Herein, the term "pharmaceutically acceptable" means that the compound is

physiologically acceptable when the compound is administered to a human, and does not cause

an allergic reaction such as a gastrointestinal disorder, dizziness or other allergic reaction, or a

systemic allergic reaction similar to these allergic reactions.

[0091] In the present disclosure, "pharmaceutically acceptable carrier" includes, but is not

limited to, binders (such as microcrystalline cellulose, alginates, gelatin and

polyvinylpyrrolidone), fillers (such as starch, sucrose, glucose and anhydrous lactic acid),

disintegrants (such as cross-linked PVP, cross-linked carboxymethyl sodium starch, croscarmellose sodium and low-substituted hydroxypropyl cellulose), lubricants (magnesium

stearate, aluminum stearate, talc, polyethylene glycol, sodium benzoate), wetting agent (such as

glycerin), surfactants (such as cetyl alcohol), and absorption enhancers, flavoring agents,

sweeteners, diluents, coating agents, etc.

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[0092] The disease as described above is one or more selected from the group consisting of

airway inflammation, asthma, bronchial asthma, allergic asthma, chronic obstructive pulmonary

disease, idiopathic pulmonary fibrosis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis,

ischemia.

[0093] A method of preventing and/or treating a disease associated with overexpression and/or

over-release of IL-17A, comprising administering the drug as described above to a subject in

need thereof.

[0094] Preferably, the above-mentioned individual is a human being, and further, the

above-mentioned individual is a human being in need of preventing and/or treating of a disease

associated with overexpression and/or over-release of IL-17A.

[0095] The disease as described above is one or more selected from the group consisting of

ischemia.

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BRIEF DESCRIPTION OF DRAWINGS

[0096] In order to more clearly illustrate the specific embodiments of the present disclosure or

the technical solutions in the conventional art, the drawings used in the specific embodiments or

the description of the conventional art will be briefly described below, and it is obvious that the

drawings in the following description are some embodiments of the present disclosure and a

person having ordinary skill in the art can obtain other drawings based on these drawings without

any creative work.

[0097] Figure 1 shows the human IL-17A binding activity of the monoclonal antibody secreted

by Clone No. 88 in Example 1;

[0098] Figure 2 shows the neutralization activity of the monoclonal antibody secreted by Clone

No. 88 in Example 1 against human IL-17A-stimulated secretion of IL-6 by HFF-1 cells;

[0099] Figure 3 shows the species specificity of the anti-human IL-17A chimeric monoclonal

antibody in Example 4;

[0100] Figure 4 shows the binding specificity of the anti-human IL-17A chimeric monoclonal

antibody in Example 4;

[0101] Figure 5 shows the in vitro neutralization activity of the anti-human IL-17A chimeric

monoclonal antibody in Example 5;

[0102] Figure 6 shows the in vivo neutralization activity of the anti-human IL-17A chimeric

monoclonal antibody in Example 6;

[0103] Figure 7 shows the concentration-time curve after the single intravenous injection in

Example 7;

[0104] Figure 8 shows the antigen binding activity of the anti-human IL-17A humanized

monoclonal antibody in Example 9;

[0105] Figure 9 shows the in vitro neutralization activity of the anti-human IL-17A humanized

monoclonal antibody in Example 10;

[0106] Figure 10 shows the concentration-time curve after the single subcutaneous injection in

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Example 15.

DETAILED DESCRIPTION

[0107] The embodiments of the present disclosure will be described in detail below with

reference to the embodiments. However, a person having ordinary skill in the art will understand

that the following embodiments are merely to illustrate present disclosure and are not intended to

limit the scope of the disclosure. For those embodiments in which specific conditions are not

specified, they were carried out according to the conventional conditions or the conditions

recommended by the manufacturer. For those used reagents or instruments of which the

manufacturers are not indicated, they were all commercially available conventional products.

Example 1. Preparation of Murine Anti-human IL-17A Monoclonal Antibody

1.1. Immunization of Animal

[0108] Female BALB/c mice, 6 to 8 weeks old, purchased from Beijing Huafukang

Biotechnology Co., Ltd., were used as experimental animals. One week after the mice were

acclimated to the environment, immunization began. The recombinant human IL-17A protein

was expressed in E. coli, and the inclusion bodies were collected and subjected to denaturation

and refolding treatment to obtain soluble IL-17A protein. For the initial immunization, 100 g of

recombinant human IL-17A-Fc protein was thoroughly mixed with Freund's complete adjuvant

(Sigma-Aldrich, Catalog Number F5881) to form an emulsion, which was intraperitoneally

injected into the mice. Two weeks later, booster immunizations were performed. For the booster

immunization, 50 g of recombinant human IL-17A-Fc protein was thoroughly mixed with

Freund's incomplete adjuvant (Sigma-Aldrich, Catalog Number F5806) to form an emulsion,

which was intraperitoneally injected into the mice. The immunization was boosted in the same

way every 2 weeks, for a total 3 times. On the seventh day after the last immunization, blood was

collected from retro orbital venous plexus of the mice and centrifuged to separate serum, and the

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antibody titer was determined by ELISA. Mice with high titers were selected for hybridization to

make hybridomas. Three days before the hybridization, 50 g of recombinant human IL-17A-Fc

protein was intraperitoneally injected into mice without adjuvant. On the day of hybridization,

the spleen was aseptically removed to prepare a single spleen cell suspension for use.

1.2. Preparation of Hybridomas

[0109] Myeloma cells SP2/0 in logarithmic growth phase were centrifuged at 1000 rpm for 5

minutes, the supernatant was discarded, and the cells were suspended in incomplete DMEM

medium (Gibco, cat No. 11965) and counted. The cells needed were taken, washed twice with an

incomplete culture medium. At the same time, a spleen cell suspension prepared from a mouse

after immunization was washed twice with an incomplete culture medium. The myeloma cells

and the spleen cells were mixed at a ratio of 1 : 10 or 1 : 5, and washed once with an incomplete

culture medium in a 50 mL plastic centrifuge tube, and then centrifuged at 1200 rpm for 8

minutes. The supernatant was discarded and a Pasteur pipette was used to remove residual liquid.

The centrifuge tube was gently tapped on palm to make the precipitated cells loose and even, and

then the tube was placed in 40 °C water bath to preheat. 1 mL of 45% PEG-4000 (pH 8.0, Sigma,

cat No. P7181) preheated to 40 °C was added with 1 mL pipette at about 1 minute (with an

optimum time of 45 seconds), stirred gently with a pipette when adding (stirred with a pipette),

visible particles should be seen with the naked eyes. 20 to 30 mL of incomplete medium

preheated to 37 °C was added to the tube with 10 mL pipette within 90 seconds to terminate PEG

action, and allowed to stand at 20 to 37 °C for 10 minutes. The tube was centrifuged at 1000 rpm

for 5 minutes, and the supernatant was discarded. 5 mL of HAT medium (DMEM + HAT, Sigma,

cat No.1 H0262-1OVL) was added, and the precipitated cells were mixed gently (remember not

to blow vigorously so as not to separate the fused cells) to make a well mixed suspension.

Additional HAT medium was added until 80 to 100 mL (the spleen cell concentration was made

to be 1 to 2x10 6/mL). The suspension was dispensed into a 96-well cell culture plate, 0.1 mL per

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well; and a 24-well plate, 1.0 to 1.5 mL per well. The plates were incubated at 37 °C incubator

with 6% CO2 . Generally, six 96-well plates were used. After 5 days, 1/2 medium was replaced

with fresh HAT medium. After 7 to 10 days, the HAT medium was replaced with HT medium

(DMEM + HT, Sigma cat No. H0137-10VL). The growth of hybridoma cells was observed

routinely, and the supernatant was collected for antibody detection after the confluence of the

cells reached 1/10 or more. The positive colonies were expanded and frozen.

1.3. Clone Screening and Identification

[0110] ELISA was used to screen anti-human IL-17A antibody from hybridoma culture

supernatants. Recombinant human IL-17A was coated on a 96-well high-absorbing ELISA plate

with a carbonate buffer solution with pH 9.6, the coating concentration was 1 g/mL, the coating

amount was 100 L per well, and the coating was carried out at 4 °C overnight. The plate was

washed five times with PBST, blocked with 300 L/well of PBST containing 1% BSA, and then

incubated at 25 °C for 1 hour. The plate was washed five times with PBST. 100 L culture

supernatant samples and the positive serum control were added to each well respectively, and

then the plated was incubated at 25 °C for 1 hour. The plate was washed five times with PBST.

Then, 100 L horseradish peroxidase-labeled anti-mouse IgG antibody (Abeam, Catalog Number

Ab7068) 1:10000 diluted in PBST containing 1% BSA was added to each well, and then the

plated was incubated at 25 °C for 1 hour. The plate was washed five times with PBST. 100

FL/well of colorimetric substrate TMB was added and incubated at room temperature for 10

minutes. Color development was terminated by adding 100 L/well of 1 M H 2 SO 4 . The

absorbance at 450 nm was read on a microplate reader. Positive clones capable of producing

anti-human IL-17A antibody were selected based on the reading value at OD 450 nm.

[0111] Whether anti-human IL-I7A antibody secreted by positive clone was a neutralizing

antibody was determined by cell assays. The anti-human IL-17A antibody sample was diluted in

DMEM complete medium (GIBCO, Catalog Number 11995-073) containing 10% FBS (Hyclone,

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Catalog Number SH30084.03). The starting concentration of the antibody was 160 nM and the

final concentration was 40 nM in the medium. The antibody was subjected to 5 fold serial

dilution, and then added to a cell culture plate, 50 L per well. 20 ng/mL of human IL-17A (final

concentration 5 ng/mL) was diluted with the same complete medium and added to the cell

culture plate at 50 L per well. The plate was incubated at 37 °C for 1 hour in an incubator with

5% CO2 . The HFF-1 cells were resuspended in complete medium and seeded into a 96-well cell

culture plate at 100 L per well, 5000 cells per well. The cells were incubated at 37 °C for 24

hours in an incubator with 5% CO 2 . After the completion of the incubation, the cell culture plate

was centrifuged at 250 X g for 5 minutes, and the culture supernatant was removed, and the

human IL-6 level was detected using human IL-6 ELISA kit (R&D systems, Catalog Number

S6050) according to the instructions. The antibody capable of inhibiting the human

IL-17A-stimulated secretion of human IL-6 by HFF-1 cells was anti-human IL-17A neutralizing

antibody. Positive clone capable of secreting anti-human IL-17A neutralizing antibody was

selected based on the strength of neutralization.

[0112] The result is shown in Figure 1. Clone No. 88 has a strong human IL-17A binding

activity. According to what is shown in Figure 2, Clone No. 88 also has pretty strong human

IL-I7A neutralization activity.

1.4. Sequencing of Monoclonal Antibody

[0113] The clones having both antigen-binding activity and antigen-neutralization activity

obtained by screening were subjected to sequencing of antibody DNA sequence. Cellular mRNA

was first extracted using RNAprep Pure Kit (Tiangen, DP430). The steps were as follows:

1x107 cells were centrifuged at 300x g for 5 minutes and collected into a centrifuge tube, and all

supernatant was carefully aspirated. The lysis step was carried out immediately. The bottom of

the centrifuge tube was flicked to loose the cell pellet, 600 L of lysis buffer RL was added with

vortex. All solution was transferred to a filtration column CS (the filtration column CS was

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placed in a collection tube), centrifuged at 12,000 rpm (-13,400x g) for 2 minutes, and the

filtrate was collected. One fold volume of 70% ethanol (usually 350 L or 600 L) was added to

the filtrate, well mixed, the obtained solution and precipitate were transferred into an adsorption

column CR3 (the adsorption column CR3 was put into a collection tube), centrifuged at 12,000

rpm (-13,400x g) for 30 to 60 seconds, the liquid waste in the collection tube was removed, the

adsorption column CR3 was put back into the collection tube. 350 L of deproteinized solution

RW1 was added to the adsorption column CR3, centrifuged at 12,000 rpm (~13,400xg) for 30 to

60 seconds, the liquid waste in the collection tube was removed, the adsorption column CR3 was

put back into the collection tube. 80 L of DNase I working solution was added to the center of

the adsorption column CR3 and the column CR3 was allowed to stand at room temperature for

15 minutes. 350 L of deproteinized solution RW1 was added to the adsorption column CR3,

centrifuged at 12,000 rpm (~13,400xg) for 30 to 60 seconds, the liquid waste in the collection

tube was removed, the adsorption column CR3 was put back into the collection tube. 500 L of

rinsing solution RW was added to the adsorption column CR3 (checked whether ethanol had

been added before use), the column CR3 was allowed to stand at room temperature for 2 minutes,

centrifuged at 12,000 rpm (~13,400x g) for 30 to 60 seconds, the liquid waste in the collection

tube was removed, the adsorption column CR3 was put back into the collection tube. The

column CR3 was centrifuged at 12,000 rpm (-13,400x g) for 2 minutes, and the waste was

removed. The adsorption column CR3 was left at room temperature for a few minutes to let the

residual rinsing solution in the adsorbent material thoroughly dry. The adsorption column CR3

was transferred into a new RNase-Free centrifuge tube, 30 to 100 L of RNase-Free ddH2 0 was

added, the tube was allowed to stand at room temperature for 2 minutes, and then centrifuged at

12,000 rpm (~13,400x g) for 2 minutes to obtain a RNA solution.

[0114] The first strand of cDNA was synthesized using the QuantScript RT kit (Tiangen,

KR103). The steps are as follows: the template RNA was thawed on ice; the primer, 10xRT mix

(containing RNasin and DTT), Super pure dNTP mixture, RNase-Free ddH2 0 were thawed at

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room temperature (15 to 25 °C), and placed on ice immediately after thawing. Each solution was

well mixed by vortexer before use, the tube was centrifuged briefly to collect residual liquid on

the side of the tube. Reverse transcription system mixture (Tiangen Bio Quant cDNA

First-Strand Synthesis Kit, Catalog Number KR103-04; lOx Reverse Transcription Buffer 2 L,

Ultra-Pure dNTP 2 L, Random Primer 2 L, Reverse Transcription Enzyme 1 L) was prepared

according to Table 1. The mixture was mixed thoroughly, the duration of vortex was no more

than 5 minutes; and then centrifuged briefly and placed on ice. Finally, the template RNA (50 ng

to 2 g) was added to the mixture, mixed thoroughly, the duration of vortex was no more than 5

seconds, centrifuged briefly to collect residual liquid on the sides of the tube, incubated at 37 °C

for 60 minutes. The first strand of cDNA produced by reverse transcription was used for

subsequent PCR reaction.

[0115] The primers used in the PCR reaction are shown in Table 1.

Table 1 PCR Primers

VH primer

F1:GAGGTGAAGCTGCAGGAGTCAGGACCTAGCCTGGTG

Ri:AGGT(C/G)(A/C)AACTGCAG(C/G)AGTC(A/T)GG

R2:AGGT(C/G)(A/C)AGCTGCAG(C/G)AGTC(A/T)GG

R3:AGGT(C/G)CAGCTGCAG(C/G)AGTC(A/T)GG

R4:CCAGGGGCCAGTGGATAGACAAGCTTGGGTGTCGTTTT

F2:ATAGACAGATGGGGGTGTCGTTTTGGC

F3:CTTGACCAGGCATCCTAGAGTCA

F4:AGGGGCCAGTGGATAGACTGATGG

F5:AGGGACCAAGGGATAGACAGATGG

R5:(G/C)A(A/G)GT(A/T/C/G)(A/C)AGCTG(G/C)AG(G/C)AGTC

R6:(G/C)A(A/G)GT(A/T/C/G)(A/C)AGCTG(G/C)AG(G/C)AGTC(A/T)GG

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VL primer

Ri:GGTGATATCGTGAT(A/G)AC(C/A)CA(G/A)GATGAACTCTC

R2:GGTGATATC(A/T)TG(A/C)TGACCCAA(A/T)CTCCACTCTC

R3:GGTGATATCGT(G/T)CTCAC(C/T)CA(A/G)TCTCCAGCAAT

F1:GGGAAGATGGATCCAGTTGGTGCAGCATCAGC

F2:GGATACAGTTGGTGCAGCATC

R4:GA(C/T)ATTGTG(A/C)T(G/C)AC(A/C)CA(A/G)(A/T)CT(A/C)CA

[0116] When primers were used, any upstream primer of the VH primers could be used with

any downstream primer; in the same way, any upstream primer of the VL primers could also be

used with any downstream primer. The target band obtained by PCR amplification was cloned

into the pGEM-T vector. A single clone was picked for DNA sequencing.

Example 2. Preparation of Chimeric Anti-human IL-17A Monoclonal Antibody

[0117] The nucleic acid sequence encoding the above mentioned antibody light chain (the

full-length of the light chain was SEQ ID NO: 7 linked to SEQ ID NO: 9) and the heavy chain

(the full-length of the heavy chain was SEQ ID NO: 8 linked to SEQ ID NO: 10) was cloned into

a eukaryotic expression vector XOGC (wherein the full-length nucleic acid sequence of the light

chain of the antibody is set forth in SEQ ID NO: 23, the full-length nucleic acid sequence of the

heavy chain of the antibody is set forth in SEQ ID NO: 24), then the expression vector was

transfected into a 293F cell line (FreeStyleTM 293-F Cells, Catalog Number R79007, invitrogen).

Cells were inoculated one day prior to transfection. Cells were harvested by centrifugation on the

day of transfection. The cells were resuspended in fresh FreeStyle TM 293 Expression Medium

(FreeStyle T M 293 Expression Medium, Catalog Number 12338001, Gibco), the cell density was

200 x 105 cells /mL. Plasmid was added according to the transfection volume, the final

concentration was 36.67 g /mL, mixed gently; then linear PEI (polyethyleneimine, linear, M.W.

25000, Catalog Number 43896, Alfa Aesar) was added, the final concentration was 55 g /mL,

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mixed gently. Thereafter, the cells were placed in a 120 rpm shaker and incubated at 37 °C for 1

hour. A 19-fold transfection volume of fresh medium was then added. Continued to incubate at

37 °C on a 120 rpm shaker. The cell culture supernatant transfected for 5 to 6 days was collected

by centrifugation.

[0118] The amino acid sequence of the light chain variable region of the antibody obtained by

PCR amplification is set forth in SEQ ID NO: 7, and the amino acid sequence of the heavy chain

variable region of antibody is set forth in SEQ ID NO: 8. The sequence of the

complementarity-determining region can be obtained by excluding the sequence of the

framework region from the mouse variable region sequence; wherein the amino acid sequences

of the three complementarity-determining regions CDR-L1, CDR-L2, CDR-L3 of the light chain

are set forth in SEQ ID NO: 1, 2 and 3, respectively; the amino acid sequences of the three

complementarity-determining regions CDR-H1, CDR-H2, CDR-H3 of the heavy chain are set

forth in SEQ ID NO: 4, 5 and 6, respectively. The amino acid sequence of the light chain

constant region of the antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the

heavy chain constant region of the antibody is set forth in SEQ ID NO: 10. The nucleic acid

sequence encoding the above mentioned antibody light chain (the full-length of the light chain

was SEQ ID NO: 7 linked to SEQ ID NO: 9) and the heavy chain (the full-length of the heavy

chain was SEQ ID NO: 8 linked to SEQ ID NO: 10) were cloned into eukaryotic expression

vector X0GC (wherein the full-length nucleic acid sequence of the light chain of the antibody is

set forth in SEQ ID NO: 23, the full-length nucleic acid sequence of the heavy chain of the

antibody is set forth in SEQ ID NO: 24), then the expression vector was transfected into 293F

cell line (FreeStyle T M 293-F Cells, Catalog Number R79007, Invitrogen). Cells were subcultured

one day prior to transfection. Cells On the day of transfection, cells were harvested by

centrifugation and then resuspended in fresh FreeStyle 293 Expression Medium (FreeStyle T M

293 Expression Medium, Catalog Number 12338001, Gibco) at a density of 200x105 cells/mL.

Plasmids were added based on the transfection volume to a final concentration of 36.67 g /mL,

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mixed gently; then linear PEI (polyethyleneimine, linear, M.W. 25000, Catalog Number 43896,

Alfa Aesar) was added to a final concentration of 55 g/mL, mixed gently. Thereafter, the cells

were placed in a shaker at 120 rpm and incubated at 37 °C for1 hour. 19-fold transfection

volume of fresh medium was then added and the cells were continually cultured at 37 °C in a

shaker at 120 rpm. The culture supernatant 5 to 6 days after transfection was collected by

centrifugation.

Example 3. Kinetics of the Binding of Chimeric Anti-human IL-17A Monoclonal Antibody

to Human IL-17A

[0119] The kinetics of the binding of anti-human IL-17A chimeric monoclonal antibody to

antigen human IL-17A was detected using Biacore 3000 Instrument. The instrument utilizes an

optical surface plasmon resonance technique to detect association and dissociation between a

molecule coupled on a sensor chip and an analyte. CM5 chips (GE Healthcare, BR-1000-12)

were used. Brief experiment procedure was as follow: human IL-17A was dissolved in sodium

acetate buffer (pH 5.0) and coupled to CM chip by injecting at a speed of 10 L/min. 1 M

ethanolamine was injected at a speed of 10 L/min for blocking. In the association phase,

different concentrations of anti-human IL-17A chimeric monoclonal antibody and control were

respectively injected at a speed of 30 L/min for 180 seconds, and during the dissociation phase,

PBS buffer was injected at a speed of 30 L/min for 600 seconds. 10 mM glycine solution (pH

2.0) was used for regeneration. Association rate constants and dissociation rate constants were

analyzed and calculated by Biacore 3000 control software. The association rate constant,

dissociation rate constant and dissociation equilibrium constant of the anti-human IL-17A

chimeric antibody are shown in Table 2. Compared with Secukinumab, the anti-human IL-17A

mAb has smaller dissociation equilibrium constant, stronger affinity, especially after binding to

IL-17A antigen, it could maintain the binding state for a longer time and is not easy to be

dissociated, which contributes to its biological functions.

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Table 2. Binding Kinetics of Anti-Human IL-17A Chimeric Antibody to Human IL-17A

Sample Detected K..(1/Ms) Krff (1/s) KD (nM)

2.52E

+ Secukinumab 7.96E - 05 0.32 05

Anti-human IL-17A Chimeric 5.41E

+ 1.49E - 06 0.03 Antibody 04

Example 4. Species Specificity and Binding Specificity of Chimeric Anti-human IL-17A

Monoclonal Antibody

[0120] The species specificity of the anti-human IL-17A chimeric monoclonal antibody was

determined by ELISA. Recombinant human IL-17A, monkey IL-17A, rat IL-17A and mouse

IL-17A (all purchased from Sino Biological Inc.), were coated on a 96-well high-absorbing

ELISA plate with a carbonate buffer solution with pH 9.6, the coating concentration was 1

ptg/mL, the coating amount was 100 L per well, and the coating was carried out at 4 °C

overnight. The plate was washed five times with PBST and blocked with 300 L/well of PBST

containing 1% BSA, and then incubated at 25 °C for 1 hour. The plate was washed five times

with PBST. The control and the anti-human IL-17A chimeric monoclonal antibody sample

serially diluted in PBST containing 1% BSA were added, 100 L per well, incubated at 25 °C for

1 hour. The plate was washed five times with PBST. Then, horseradish peroxidase-labeled

anti-human IgG antibody (Chemicon, Catalog Number AP309P) 1:2000 diluted in PBST

containing 1% BSA was added, 100 L per well, incubated at 25 °C for 1 hour. The plate was

washed five times with PBST. 100 L/well of colorimetric substrate TMB was added and

incubated at room temperature for 10 minutes. Color development was terminated by adding 100

pL/well of 1 M H 2 SO 4 . The absorbance at 450 nm was read on a microplate reader.

[0121] The binding specificity of the anti-human IL-17A chimeric monoclonal antibody was

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determined by ELISA. Recombinant human IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-I, IL-2, IL-6, IL-8, IL-21, IL-22, IL-23, IFN-g and TNFa (purchased from Sino Biological Inc. or R&D systems) were coated on a 96-well high-absorbing ELISA plate with a carbonate buffer solution with pH 9.6, the coating concentration was 1 g/mL, the coating amount was 100 L per well, and the coating was carried at 4 °C out overnight. The plate was washed five times with PBST and blocked with 300 L/well of PBST containing 1% BSA and incubated at 25 °C for 1 hour. The plate was washed five times with PBST. The control and the anti-human IL-17A chimeric monoclonal antibody sample diluted in PBST containing 1% BSA were added, 100 L per well, incubated at 25 °C for 1 hour. The plate was washed five times with PBST. Then, horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, Catalog Number AP309P) 1:2000 diluted in PBST containing 1% BSA was added, 100 L was added to each well, incubated at 25 °C for 1 hour. The plate was washed five times with PBST. 100 L/well of colorimetric substrate TMB was added and incubated at room temperature for 10 minutes. Color development was terminated by adding 100 L/well of 1 M H2 SO 4 . The absorbance at 450 nm was read on a microplate reader.

[0122] The binding specificity of the anti-human IL-17A chimeric monoclonal antibody was determined by ELISA. Recombinant human IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, IL-I, IL-2, IL-6, IL-8, IL-21, IL-22, IL-23, IFN-g and TNFa (purchased from Sino Biological Inc. or R&D systems) were coated on a 96-well high-absorbing ELISA plate with a carbonate buffer solution with pH 9.6, the coating concentration was 1 g/mL, the coating amount was 100 L per well, and the coating was carried at 4 °C out overnight. Washed five times with PBST. Blocked with 300 L/well of PBST containing 1% BSA and incubated at 25 °C for 1 hour. Washed five times with PBST. The control and the anti-human IL-17A chimeric monoclonal antibody sample diluted in PBST containing 1% BSA were added, 100 L was added to each well, incubated at 25 °C for 1 hour. Washed five times with PBST. Then, horseradish peroxidase-labeled anti-human IgG antibody (Chemicon, Catalog Number AP309P) 1:2000

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diluted in PBST containing 1% BSA was added, 100 L was added to each well, incubated at 25 °C for 1 hour. Washed five times with PBST. 100 L/well of colorimetric substrate TMB was added, developed at room temperature for 10 minutes. Color development was terminated by adding 100 L/well of 1 M H 2 SO 4 . The absorbance at 450 nm was read on a microplate reader.

[0123] The result is shown in Figure 3. The anti-human IL-17A chimeric monoclonal antibody bound to human and monkey IL-17A, but not rat or mouse IL-17A, indicating the antibody was species-specific. In addition, as shown in Figure 4, the anti-human IL-17A chimeric monoclonal antibody also has strong binding specificity, which only bound to IL-17A but not other cytokines of IL-17 family or unrelated cytokines.

Example 5. Neutralization Activity of Chimeric Anti-human IL-17A Monoclonal Antibody In Vitro

[0124] The anti-human IL-17A antibody sample was diluted in DMEM complete medium

(GIBCO, Catalog Number 11995-073) containing 10% FBS (Hyclone, Catalog Number SH30084.03). The starting concentration of the antibody was 160 nM and the final concentration was 40 nM in the medium. The antibody was subjected to 5 fold serial dilution, and then added to a cell culture plate, 50 L per well. 20 ng/mL of human IL-17A (final concentration 5 ng/mL) was diluted with the same complete medium and added to the cell culture plate at 50 L per well. The plate was incubated at 37 °C for 1 hour in an incubator with 5% CO2 . The HFF-1 cells were resuspended in complete medium and seeded into a 96-well cell culture plate at 100 L per well, 5000 cells per well. The cells were incubated at 37 °C for 24 hours in an incubator with 5% CO 2 .

After the completion of the incubation, the cell culture plate was centrifuged at 250 X g for 5 minutes, and the culture supernatant was removed, and the human IL-6 level was detected using human IL-6 ELISA kit (R&D systems, Catalog Number S6050) according to the instructions.

[0125] The result is shown in Figure 5. Compared to Secukinumab, the anti-human IL-17A chimeric monoclonal antibody has a stronger IL-17A neutralization activity in vitro, and a better

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effect on inhibiting the human IL-17A-stimulated secretion of human IL-6 by HFF-1 cells.

Example 6. Neutralization Activity of Chimeric Anti-human IL-17A Monoclonal Antibody

In Vivo

[0126] Female BALB/c mice, 6 to 8 weeks old, purchased from Beijing Huafukang

acclimated to the environment, the mice were randomly divided into groups, 6 per group. Each

group was given anti-human IL-17A chimeric monoclonal antibody or control monoclonal

antibody Secukinumab, at three doses of 0.7 nmol/kg, 7 nmol/kg or 70 nmol/kg, intravenous

injection, single administration. One hour after the administration, human IL-17A was injected

subcutaneously, 10 g per mouse. Two hours later, retro-orbital blood sample was collected

without anticoagulation, and the blood sample was allowed to stand at room temperature for 30

minutes to 1 hour. After the coagulation of blood, the sample was centrifuged at 3000 rpm for 10

minutes to obtain a serum sample. The concentration of mouse CXCL1 (C-X-C Motif

Chemokine Ligand, also known as KC) in the serum was detected according to the instructions

using a mouse CXCL1 ELISA kit (RayBiotech, Catalog Number ELM-KC).

[0127] The result is shown in Figure 6. The anti-human IL-17A chimeric monoclonal antibody

inhibited the secretion of CXCL1 in human IL-17A-stimulated mice, showing a stronger activity

than the control monoclonal antibody Secukinumab.

Example 7. Pharmacokinetics Study of Chimeric Anti-Human IL-17A Monoclonal

Antibody in Rats

[0128] Female SD rats, 6 to 8 weeks old, purchased from Beijing Huafukang Biotechnology

Co., Ltd., were used as experimental animals. One week after the rats were acclimated to the

environment, the rats were randomly divided into groups, 3 rats per group. Anti-human IL-17A

chimeric monoclonal antibody and control monoclonal antibody Secukinumab were

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administered respectively at a dose of 20 nmol/kg by intravenous injection, single dose. At 0, 5

minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours,

168 hours, 216 hours, 264 hours, 312 hours after administration, the retro-orbital blood sample

was collected without anticoagulation, and the blood sample was allowed to stand at room

temperature for 30 minutes to 1 hour; after coagulation, the blood sample was centrifuged at

3,000 rpm for 10 minutes, the obtained serum sample was frozen at -80 °C and stored for testing.

[0129] The concentrations of anti-human IL-17A chimeric monoclonal antibody and control

monoclonal antibody Secukinumab in the serum were determined by ELISA. Briefly, human

recombinant IL-17A protein was coated on a high-absorbing ELISA plate with a carbonate

buffer solution with pH 9.6 at 4 °C overnight. The plate was washed with PBST. To prevent

non-specific binding, the plate was blocked with PBST containing 5% nonfat milk powder, and

then washed with PBST. Then, the serum sample to be tested diluted with PBST containing 10%

mixed rat serum and 1% BSA was added and incubated at 25 °C for 1 hour, and the plate was

washed with PBST. Horseradish peroxidase-labeled anti-human IgG antibody (Chemicon,

Catalog Number AP309P) diluted in PBST containing 5% skimmed milk powder was added,

incubated at 25 °C for 1 hour, the then plate was washed with PBST. Finally, color development

was carried out using the colorimetric substrate TMB at room temperature for 10 minutes. Color

development was terminated by adding 100 pL/well of 1 M H2 SO 4 . The absorbance at 450 nm

was read on a microplate reader.

[0130] The result is shown in Figure 7. A single intravenous injection dose of 20 nmol/kg of

anti-human IL-17A chimeric monoclonal antibody or control monoclonal antibody Secukinumab

showed similar concentration-time curves and pharmacokinetic features in rats. The

pharmacological parameters of the anti-human IL-17A chimeric monoclonal antibody are as

follows: half-life t 1 2 was 458 hours; the area under the concentration-time curve AUCiast was

44,286 nM.hr; the estimated initial concentration Co was 413 nM; the apparent volume of

distribution Vd was 115 mL/kg, the clearance rate CL was 0.17 mL/hr/kg; the mean residence

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time MRTias was 140 hours.

Example 8. Preparation of Humanized Anti-Human IL-17A Monoclonal Antibody

[0131] The humanized form of the anti-IL-17 antibody was obtained according to the method

of Leung et al. (1995, Molecule Immunol 32: 1413-27).

[0132] The humanized template that best matches murine non-CDR region was selected from

Germline database. The template for the heavy chain variable region was IGVH4-59*01 and the

sequence is set forth in SEQ ID NO: 35. The template for the light chain variable region was

IGKV2-30*02 and the sequence is set forth in SEQ ID NO: 36. The CDR region of murine

antibody was grafted onto the selected humanized template, replacing the CDR region of the

human template. The obtained grafted humanized antibody heavy chain variable region has a

sequence set forth in SEQ ID NO: 37, and the grafted humanized antibody light chain variable

region has a sequence set forth in SEQ ID NO: 38. Nine positions selected by sequence

alignment were subjected to back mutations, including 4 positions on heavy chains: L4V,149M,

V681, V72R, and 5 positions on light chains: D1I, V21, F41Y, R51L, Y92F. Different humanized

sequences were constructed by reducing the number of back mutations, and the heavy chain

sequences and the light chain variable region sequences are shown in Table 3. The heavy chain

variable region (SEQ ID NO: 27-34) of the humanized anti-human IL-17A monoclonal antibody

was linked to the heavy chain constant region (SEQ ID NO: 10) of human antibody IgG1 to

obtain corresponding full-length sequence of heavy chain. The light chain variable region (SEQ

ID NO: 19-26) of the humanized anti-human IL-17A monoclonal antibody was linked to the light

chain constant region (SEQ ID NO: 9) of the human Kappa antibody to obtain corresponding

full-length sequence of light chain. The full-length sequence of the heavy chain was combined

with the full-length sequence of the light chain to obtain a full-length sequence of the humanized

antibody. The full-length sequence was digested with EcoRI and HindlIl, and then inserted into

XOGC vector.

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Table 3. The Sequences of the Heavy Chain Variable Region and Light Chain Variable Region of

Anti-human IL-I7A Humanized Antibody

VH SEQIDNO:

Grafted 27

BM 28

AS15799 29

AS15802 30 AS15803 31

AS15805/ 32

AS15815

AS15810 33

AS15820 34

VL SEQIDNO:

Grafted 19

BM 20

AS15799 21

AS15802/AS15803 22

AS15805 23

AS15810 24

AS15815 25

AS15820 26

Example 9. Antigen Binding Activity of Humanized Anti-human IL-17A Monoclonal

Antibody

[0133] The antigen binding activity of humanized anti-human IL-17A monoclonal antibody

was determined by ELISA. Recombinant human IL-17A (purchased from Sino Biological Inc.)

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was coated on a 96-well high-absorbing ELISA plate with a carbonate buffer solution with pH

9.6, the coating concentration was 1 g/mL, the coating amount was 100 L per well, and the

coating was carried at 4 °C out overnight. The plate was washed five times with PBST and

blocked with 300 L/well of PBST containing 1% BSA and incubated at 25 °C for 1 hour. The

plate was washed five times with PBST. The control and the anti-human IL-17A chimeric

monoclonal antibody sample diluted in PBST containing 1% BSA were added, 100 L per well, incubated at 25 °C for 1 hour. The plate was washed five times with PBST. Then, horseradish

peroxidase-labeled anti-human IgG antibody (Chemicon, Catalog Number AP309P) 1:2000

diluted in PBST containing 1% BSA was added, 100 L was added to each well, incubated at

25 °C for 1 hour. The plate was washed five times with PBST. 100 L/well of colorimetric

substrate TMB was added and incubated at room temperature for 10 minutes. Color development

was terminated by adding 100 L/well of 1 M H 2 SO 4 . The absorbance at 450 nm was read on a

microplate reader.

[0134] The result is shown in Figure 8. All of the anti-human IL-7A humanized monoclonal

antibodies AS15799, AS15802, AS15803, AS15805, AS15810, AS15815 and AS15820 could

bind human IL-I7A with a high affinity activity.

Example 10. Neutralization Activity of Humanized Anti-human IL-17A Monoclonal

AntibodyIn Vitro

[0135] The anti-human IL-17A antibody sample was diluted in DMEM complete medium

(GIBCO, Catalog Number 11995-073) containing 10% FBS (Hyclone, Catalog Number

SH30084.03). The starting concentration of the antibody was 160 nM and the final concentration

was 40 nM in the medium. The antibody was subjected to 5 fold serial dilution, and then added

to a cell culture plate, 50 L per well. 20 ng/mL of human IL-17A (final concentration 5 ng/mL)

was diluted with the same complete medium and added to the cell culture plate at 50 L per well.

The plate was incubated at 37 °C for 1 hour in an incubator with 5% CO2 . The HFF-1 cells were

English Translation of PCT/CN2017/101083

resuspended in complete medium and seeded into a 96-well cell culture plate at 100 L per well,

5000 cells per well. The cells were incubated at 37 °C for 24 hours in an incubator with 5% CO 2

. After the completion of the incubation, the cell culture plate was centrifuged at 250 X g for 5

minutes, and the culture supernatant was removed, and the human IL-6 level was detected using

human IL-6 ELISA kit (R&D systems, Catalog Number S6050) according to the instructions.

[0136] The result is shown in Figure 9. Compared to Secukinumab, anti-human IL-17A

humanized monoclonal antibodies AS15799, AS15802, AS15803, AS15805, AS15810, AS15815

and AS15820 have stronger IL-I7A neutralization activity and stronger effect on inhibiting the

human IL-17A-stimulated secretion of human IL-6 by HFF-1 cells in vitro.

Example 11. Detection of Purity and Thermal Stability of Humanized Anti-human IL-17A

Monoclonal Antibody by Size-exclusion High-performance Liquid Chromatography

(SE-HPLC)

[0137] TSKgel SuperSW3000 chromatography column (Catalog Number: 0018675) was used.

The mobile phase was 0.mol/1 of phosphate buffer (NaH 2PO 4 -Na2HPO4), 0.1 mol/1 of sodium

sulfate buffer, pH 6.7; the flow rate was 0.35 mL/min; the column temperature was 25 °C;

sample pool temperature was 4 °C; detection wavelength was 280 nm. The sample was diluted

with sample buffer to 1 mg/mL, and the injection volume was 5 L. The experiment result was

processed by Agilent High Performance Liquid Chromatograph 1260 System Workstation, and

purity was calculated by the percentage of the main peak using area normalization method. The

humanized anti-human IL-17A monoclonal antibody prepared above was subjected to SE-HPLC

purity assay. To determine the thermal stability of these monoclonal antibodies, the samples

were placed under high temperature conditions of 40 °C, and the samples were subjected to

SE-HPLC assay at week 2 and week 4 respectively to observe thermal stability, and the result is

shown in Table 4 below. All of the humanized anti-human IL-17A antibodies showed good and

considerable stability.

English Translation of PCT/CN2017/101083

Table 4. Thermal Stability of Humanized Anti-human IL-17A Monoclonal Antibody at 40 °C by

SE-HPLC

SE-HPLC Purity Humanized Anti-human IL-I7A Monoclonal Antibody T=O Week 2 Week 4

AS15802 99.9% 98.5 97.3

AS15803 99.9% 98.3 96.9

AS15810 98.0% 96.9 95.9 AS15815 98.1% 97.2 96.5

AS15820 96.5% 95.7 94.5

Example 12. Determination of Tm Value of Humanized Anti-human IL-17A Monoclonal

Antibody

[0138] The melting temperature (Tm) of the humanized anti-human IL-17A monoclonal

antibody was determined by Differential Scanning Fluorimetry (DSF). DSF is a method for

detecting the thermal denaturation process of proteins in a sample by using the fluorescence

intensity change of the fluorescent indicator to determine the protein denaturation temperature.

The reagent used was SYPRO Orange Protein Fluorescent Dye (Sigma-Aldrich, USA, Catalog

Number S5692; 5000x concentration, in DMSO). AB 7500 Real Time PCR machine was

purchased from Applied Biosystems, Inc., USA. The protein fluorescent dye was diluted 1:50

with sample buffer, and 1 L of the diluted dye was mixed with 19 L of protein solution, so the

final dilution of the fluorescent dye was 1:1000. The diluted fluorescent dye was added to a

96-well plate, and three parallel wells were set for each sample. The plate was sealed with an

optical sealing film, centrifuged at 1000 rpm for 2 minutes to remove air bubbles. The RT-PCR

program was set as follows: melting curve was set in continuous mode, scanning temperature

range was 25 to 99 °C, heating rate was 1% (about 1 C/min), and then 25 °C for 2 min. Data

was collected during heating, the reporter group was set as "ROX", the quenching group was set

English Translation of PCT/CN2017/101083

as "None", and the reaction volume was 20 pL. The sample concentration was 1 mg/mL, and the

reference solution was sample buffer. Fluorescence curves and the first derivative were plotted

using Protein Thermal ShiftTM Software v1.3 software. In the DSF test, the midpoint temperature

of the first transition of the protein is usually considered as the denaturation temperature of the

thermal stability of the protein. The Tm values of the humanized anti-human IL-17A monoclonal

antibody were measured and the result is shown in Table 5 below. All of the humanized

anti-human IL-17A monoclonal antibodies have pretty good Tm value.

Table 5. Tm Value of Humanized Anti-human IL-17A Monoclonal Antibody

Humanized Anti-human IL-I7A Monoclonal Antibodies Tm Value

AS15802 67.2 °C

AS15803 68.3 °C AS15810 67.9 °C

AS15815 69.3 °C

AS15820 67.7 °C

Example 13. Detection of Charge Isomers of Humanized Anti-Human IL-17A Monoclonal

Antibody by Cation Exchange Chromatography (CEX)

[0139] Cation exchange chromatography column MabPac SCX-10 was used, 4 mm x 250 mm

(Catalog Number: 78655). 20 mmol/L of 2-(N-morpholine) ethanesulfonic acid (MES) (pH 5.6)

and 60 mmol/L of sodium chloride were used as mobile phase A; 20 mmol/L of MES (pH 5.6)

and 300 mmol/L of sodium chloride were used as mobile phase B. The flow rate was 0.5 mL/min;

the column temperature was 25 0 C; sample pool temperature was 4 °C; detection wavelength was

280 nm; the sample loading volume was 50 pL (1 mg/mL); the elution was carried out in a linear

gradient from 5 to 50% over 60 minutes. The experiment result was processed by Agilent High

Performance Liquid Chromatograph 1260 System Workstation, and the percentage of the peak

area was calculated by the area normalization method. The humanized anti-human IL-17A

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monoclonal antibodies were subjected to CEX detection. To determine the chemical stability of

these monoclonal antibodies, the above samples were put under high temperature conditions of

40 °C, and the samples were taken out at week 2 and week 4 respectively for CEX detection and

the changes in the proportion of charge variants was observed. The result is shown in Table 6.

All of the humanized anti-human IL-17A antibodies have a relatively low proportion of charge

variants.

Table 6. Changes in Charge Variants of Humanized Anti-human IL-I7A Monoclonal Antibody at 40 °C by CEX

Changes in Charge Variants

T=O Week 2

AS15802 64.9 15.5 19.6 53.5 32.7 13.8

AS15803 61.8 17.5 20.7 51.7 37.5 10.8

AS15810 59.7 14 26.3 49.9 36.7 13.5

AS15815 60.3 14.4 25.3 47 40.4 12.6

AS15820 58.7 16.6 24.7 49.1 35.6 15.3

Example 14. Stability of Humanized Anti-human IL-17A Monoclonal Antibody under Low

pH Condition for Virus Inactivation

[0140] 200 g of the test sample was adjusted to pH 3.4 0.05 with 1 mol/ citric acid mother

solution, and the final concentration of the sample was 1 mg/mL. The sample was left at room

temperature and sampled at 0, 1, 2, 4 and 6 hours respectively, and the pH was adjusted to 7.5

with 2 mol/l of Tris-HCl pH 9.5 mother solution. The samples were analyzed by the above SEC

method, and the result is shown in Table 7. The humanized anti-human IL-17A antibody could

tolerant low pH condition for virus inactivation for at least 6 hours, indicating a good stability.

Table 7. Stability of Humanized Anti-human IL-I7A Monoclonal Antibody under Low pH

Condition detected by SE-HPLC

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Humanized Anti-human SE-HPLC Purity

IL-17A Monoclonal T=O 1" Hour 2"d Hour 4th Hour 6th Hour Antibody

AS15802 99.9% 99.8 99.9 99.9 99.8

AS15803 99.9% 99.8 99.8 99.9 99.9

AS15810 98.0% 98.1 98.1 98.0 98.2

AS15815 98.1% 98.4 98.3 98.3 98.5

AS15820 96.5% 96.4 96.4 96.5 96.7

Example 15 Pharmacokinetics Study of Humanized Anti-Human IL-17A Monoclonal

Antibody in Rats

[0141] Female SD rats, 6 to 8 weeks old, purchased from Beijing Huafukang Biotechnology

chimeric monoclonal antibody and control antibody were administered respectively at a dose of

15 nmol/kg by subcutaneous injection, single administration. At 0, 1 hour, 4 hours, 8 hours, 24

hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 216 hours, 264 hours, 312 hours, 360

hours, 408 hours and 480 hours after administration, the retro-orbital blood sample was collected

minutes to 1 hour; after coagulation, the blood sample was centrifuged at 3,000 rpm for 10

minutes, the obtained serum sample was frozen at -80 °C and stored for testing.

[0142] The concentrations of anti-human IL-17A chimeric monoclonal antibody and control

antibody in the serum were determined by ELISA. Briefly, human recombinant IL-17A protein

was coated on a high-absorbing ELISA plate with a carbonate buffer solution with pH 9.6 at

4 °C overnight. The plate was washed with PBST. To prevent non-specific binding, the plate was

blocked with PBST containing 5% nonfat milk powder, and then washed with PBST. Then, the

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serum sample to be tested diluted with PBST containing 10% mixed rat serum and 1% BSA was

added and incubated at 25 °C for 1 hour, and the plate was washed with PBST. Horseradish

peroxidase-labeled anti-human IgG antibody (Chemicon, Catalog Number AP309P) diluted in

PBST containing 5% skimmed milk powder was added, incubated at 25 °C for 1 hour, the then

plate was washed with PBST. Finally, color development was carried out using the colorimetric

substrate TMB at room temperature for 10 minutes. Color development was terminated by

adding 100 L/well of 1 M H 2 SO 4 . The absorbance at 450 nm was read on a microplate reader.

[0143] The result is shown in Figure 10. The single subcutaneous injection of 15 nmol/kg of

different anti-human IL-17A humanized monoclonal antibody, control monoclonal antibody

Secukinumab, and control monoclonal antibody Ixekizumab showed similar concentration-time

curves and pharmacokinetic features in vivo.

[0144] Finally, it should be understood that the above embodiments are only used to illustrate

the technical solution of the present disclosure instead of limiting it; although the present

disclosure has been described in detail with reference to the foregoing embodiments, it should be

understood by those having ordinary skill in the art that the technical solutions described in the

foregoing embodiments may be modified, or some or all of the technical features may be

equivalently replaced; and the modifications or replacements, however, would not make the

substances of the corresponding technical solutions depart from the scope of the technical

solutions of the embodiments of the present disclosure.

[0145] Industrial Applicability: the antibody and functional fragment thereof provided by the

present disclosure can specifically bind to IL-17A, and can be used for prevention and/or

treatment of a disease associated with overexpression and/or over-release of IL-I7A, for example,

English Translation of PCT/CN2017/101083

ischemia.

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SEQUENCE LISTING

<110> BEIJING HANMI PHARM. CO., LTD.

<120> ANTIBODY SPECIFICALLY BINDING TO IL-17A AND FUNCTIONAL FRAGMENT THEREOF

<150> CN 201610827097.2 <151> 2016-09-14

<150> PCT/CN2017/101083 2017328310

<151> 2016-09-08

<160> 38

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Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45

Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 2017328310

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95

Ala Arg Glu Glu Tyr Asp Asp Ile Tyr Ala Val Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Ser Val Thr Val Ser Ser 115 120

<210> 29 <211> 120 <212> PRT <213> Artificial

<400> 29

Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Page 15

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65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Glu Tyr Asp Asp Ile Tyr Ala Val Asp Tyr Trp Gly Gln 100 105 110 2017328310

Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 30 <211> 120 <212> PRT <213> Artificial

<400> 30

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Glu Tyr Asp Asp Ile Tyr Ala Val Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120 Page 16

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<210> 31 <211> 120 <212> PRT <213> Artificial

<400> 31

Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 2017328310

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Glu Tyr Asp Asp Ile Tyr Ala Val Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 32 <211> 120 <212> PRT <213> Artificial

<400> 32

Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

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Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Glu Tyr Asp Asp Ile Tyr Ala Val Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 33 <211> 120 <212> PRT <213> Artificial

<400> 33

Gln Val Gln Val Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Page 18

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65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Gly Thr Leu Val Thr Val Ser Ser 115 120

<210> 34 <211> 120 <212> PRT <213> Artificial

<400> 34

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

Met Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Glu Tyr Asp Asp Ile Tyr Ala Val Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120 Page 19

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<210> 35 <211> 98 <212> PRT <213> Artificial

<220> <221> misc_feature 2017328310

<222> (98)..(98) <223> Xaa can be any naturally occurring amino acid

<400> 35

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Xaa

<210> 36 <211> 101 <212> PRT <213> Artificial

<400> 36

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Page 20

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Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 2017328310

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95

Thr His Trp Pro Pro 100

<210> 37 <211> 120 <212> PRT <213> Artificial

<400> 37

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asn Ser Ile Thr Ser Tyr 20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

Ile Gly Tyr Ile Thr Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80

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Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Glu Glu Tyr Asp Asp Ile Tyr Ala Val Asp Tyr Trp Gly Gln 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 2017328310

115 120

<210> 38 <211> 111 <212> PRT <213> Artificial

<400> 38

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Tyr Asn Arg Phe Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95

Thr His Phe Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110

Page 22

Claims

The claims defining the invention are as follows:

1. An antibody capable of specifically binding to IL-17A or a functional fragment capable of

specifically binding to IL-17A thereof, wherein the antibody or the functional fragment thereof

comprise a light chain and a heavy chain;

the light chain comprises a light chain CDR consisting of CDR-L1, CDR-L2 and CDR-L3;

the heavy chain comprises a heavy chain CDR consisting of CDR-H1, CDR-H2 and CDR-H3;

the amino acid sequences of the CDR-L1, CDR-L2 and CDR-L3 are respectively set forth in

SEQ ID NO: 1, 2 and 3; the amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 are

respectively set forth in SEQ ID NO: 4, 5 and 6;

preferably, the antibody or the functional fragment thereof includes an IL-17A chimeric

antibody or a functional fragment thereof, and an IL-17A humanized antibody or a functional

fragment thereof.

2. The antibody or the functional fragment thereof according to claim 1, wherein the antibody

comprises a constant region sequence of any one selected from the group consisting of human

IgGI, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

3. The antibody or the functional fragment thereof according to claim 1 or 2, wherein the

functional fragment comprises one or more selected from the group consisting of F(ab')2, Fab',

Fab, Fv, scFv, bispecific antibody and antibody minimal recognition unit.

4. The antibody or the functional fragment thereof according to any one of claims 1 to 3,

of the IL-17A chimeric antibody or the functional fragment thereof are respectively set forth in

SEQ ID NO: 7 and SEQ ID NO:8; preferably, the amino acid sequence of the light chain constant region and the heavy chain constant region of the IL-17A chimeric antibody or functional fragment thereof are respectively set forth in SEQ ID NO: 9 and SEQ ID NO: 10.

5. The antibody or the functional fragment thereof according to any one of claims 1 to 3,

wherein light chain framework region of the IL-17A humanized antibody or the functional

region of the IL-17A humanized antibody or the functional fragment thereof comprises FR-Hi,

FR-H2, FR-H3 and FR-H4;

the FR-Li is selected from the amino acid sequence set forth in SEQ ID NO: I Iand the

amino sequence having the following substitution or a combination thereof:

the I1 amino acid D is replaced by I;

the 2"d amino acid V is replaced by I;

the FR-L2 is selected from the amino acid sequence set forth in SEQ ID NO: 12 and the

amino sequence having the following substitution or a combination thereof:

the 4 th amino acid F is replaced by Y;

the 14th amino acid R is replaced by L;

the FR-L3 is selected from the amino acid sequence set forth in SEQ ID NO: 13 and the

amino sequence having the following substitution or a combination thereof:

the 3 5 th amino acid Y is replaced by F;

the FR-L4 is selected from the amino acid sequence set forth in SEQ ID NO: 14;

the FR-Hi is selected from the amino acid sequence set forth in SEQ ID NO: 15 and the

amino sequence having the following substitution or a combination thereof:

the 4 th amino acid L is replaced by V;

the FR-H2 is selected from the amino acid sequence set forth in SEQ ID NO: 16 and the

amino sequence having the following substitution or a combination thereof:

English Translation of PCT/CN2017/101083

the 1 5 th amino acid I is replaced by M;

the FR-H3 is selected from the amino acid sequence set forth in SEQ ID NO: 17 and the

amino sequence having the following substitution or a combination thereof:

the 2"d amino acid V is replaced by I;

the 6 th amino acid V is replaced by R;

the FR-H4 is selected from the amino acid sequence set forth in SEQID NO: 18;

preferably, light chain variable region sequence of the IL-17A humanized antibody or the

functional fragment thereof is one selected from SEQ ID NO: 19-26;

preferably, heavy chain variable region sequence of the IL-17A humanized antibody or the

functional fragment thereof is one selected from SEQ ID NO: 27-34;

more preferably, the light chain variable region sequence of the IL-17A humanized antibody

or the functional fragment thereof is set forth in SEQ ID NO: 19; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 27;

alternatively, the light chain variable region sequence of the IL-17A humanized antibody or

the functional fragment thereof is set forth in SEQ ID NO: 20; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 28;

the functional fragment thereof is set forth in SEQ ID NO: 21; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 29;

the functional fragment thereof is set forth in SEQ ID NO: 22; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 30;

variable region sequence is set forth in SEQ ID NO: 31;

English Translation of PCT/CN2017/101083

the functional fragment thereof is set forth in SEQ ID NO: 23; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 32;

the functional fragment thereof is set forth in SEQ ID NO: 24; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 33;

the functional fragment thereof is set forth in SEQ ID NO: 25; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 32;

the functional fragment thereof is set forth in SEQ ID NO: 26; the corresponding heavy chain

variable region sequence is set forth in SEQ ID NO: 34;

more preferably, the amino acid sequences of the light chain constant region and the heavy

chain constant region of the IL-17A humanized antibody or the functional fragment thereof are

respectively set forth in SEQ ID NO: 9 and SEQ ID NO: 10.

6. An isolated nucleic acid molecule selected from:

A) DNA or RNA, encoding the antibody or the functional fragment thereof according to any

one of claims I to 5; and

B) a nucleic acid complementary to the nucleic acid as defined in A).

7. A composition, comprising the antibody and/or the functional fragment thereof according to

any one of claims 1 to 5, or a compound of the antibody and/or the functional fragment thereof

together with other components, as an active ingredient.

English Translation of PCT/CN2017/101083

8. The composition according to claim 7, wherein the antibody or the functional fragment thereof are coupled to at least one diagnostic agent and/or therapeutic agent to form an immunoconjugate.

9. The composition according to claim 8, wherein the diagnostic agent is one or more selected from the group consisting of a radionuclide, a radioactive contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photosensitizer; preferably, the radionuclide is one or more selected from the group consisting of ""In, "IIn, "7Lu, 18F, 52Fe, 62Cu, 14Cu, 67Cu, 67Ga, 68Ga, 6Y, 9Y, 89Zr, 94mTc, 94Tc, 99mTc, 120 1,23 124 1, 25,

131 is4-issGd, 32P, C, 1N, o, 18 Re, Re, 5Mn, 52mMn, 5Co, 72As, 7Br, 76Br, 82 mRb and 83Sr

preferably, the paramagnetic ion is one or more selected from the group consisting of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III); preferably, the fluorescent label is one or more selected from the group consisting of Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, 5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,

6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl violet, brilliant cresyl blue, 4-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate,

English Translation of PCT/CN2017/101083

tri-bipyridyldiamine europium, europium cryptate compound or chelate, diamine, dicyanine, La

Jolla blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine,

R-phycoerythrin, C-Phycocyanin, phycoerythrin R, REG, rhodamine green, rhodamine

isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol),

tetramethylrhodamine and Texas Red.

10. The composition according to claim 8 or 9, wherein the therapeutic agent is one or more

selected from the group consisting of a naked antibody, a cytotoxic agent, a drug, a radionuclide,

a boron atom, an immunomodulator, an anti-apoptotic agent, a photosensitizing therapeutic, an

immunoconjugate and an oligonucleotide;

preferably, the drug is one or more selected from the group consisting of dexamethasone,

diclofenac, nabumetone, meloxicam, celecoxib, methotrexate, cyclophosphamide, azathioprine,

cyclosporine A and vincristine;

preferably, the oligonucleotide is one or more selected from the group consisting of shRNA,

miRNA and siRNA;

preferably, the immunomodulator is one or more selected from the group consisting of a

stimulating factor (GM-CSF) and stem cell growth factor;

preferably, the radionuclide is one or more selected from the group consisting of .."In, "At, 17 7 Lu, 211Bi, 2 12 Bi, 2 13 Bi, 211At, 62 CU, 67 CU, 90 Y 125 1,131 1,133 1, 32 P 33 P 47Sc, IAg, 67Ga, 153Sm,

16 1 11 152 166 161 166 186 188 189 211 1 22112 2223 25 77 89 99 Tb, Dy, Dy, Ho, Ho, Re, Re, Re, Pb, Pb, Ra, Ac, As, Sr, Mo, 15Rh, 149Pm, 169Er, 194Ir, Co, 80mBr, 99mTc, 103mRh, 19Pt, 119Sb, 189mOs, 192 219Rn, 21Po,

221Fr, 25sFm, "C, 1N, o, 75Br, 198Au, 199Au, 224Ac, 7Br, "3mIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg,

203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 142Pr, 143Pr, 16 1 Tb, Co, 58Co,

English Translation of PCT/CN2017/101083

5Cr, 59Fe, 7Se, 201TI, 7'Br and "9Yb.

11. Use of the composition according to any one of claims 7 to 10 for the manufacture of a

medicament in prevention and/or treatment of a disease associated with overexpression and/or

over-release of IL-17A;

preferably, the disease is one or more selected from the group consisting of airway

idiopathic pulmonary fibrosis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus,

ischemia.

12. Use of the antibody capable of specifically binding to IL-17A or the functional fragment

capable of specifically binding to IL-17A thereof according to any one of claims 1 to 5 for the

manufacture of a medicament in prevention and/or treatment of a disease associated with

overexpression and/or over-release of IL-17A;

Crohn's disease, allograft rejection, Behcet's disease, sepsis, septic or endotoxin shock

and ischemia.

13. A drug for prevention and/or treatment of a disease associated with

overexpression and/or over-release of IL-17A, comprising the antibody capable of

specifically binding to IL-17A or the functional fragment capable of specifically

binding to IL-17A thereof according to any one of claims 1 to 5, and a

pharmaceutically acceptable carrier;

alternatively, the drug comprises the composition according to any one of claims

7-10 and a pharmaceutically acceptable carrier;

preferably, the disease is one or more selected from the group consisting of

airway inflammation, asthma, bronchial asthma, allergic asthma, chronic obstructive

pulmonary disease, idiopathic pulmonary fibrosis, rheumatoid arthritis, osteoarthritis,

ankylosing spondylitis, psoriatic arthritis, psoriasis, multiple sclerosis, systemic

sclerosis, systemic lupus erythematosus, lupus nephritis, scleroderma, ulcerative

colitis, inflammatory bowel disease, uveitis, helicobacter pylori-associated gastritis,

osteoporosis, bone erosion, intraperitoneal abscess and adhesions, Addison's disease,

gamma globulin deficiency, alopecia areata, celiac disease, Chagas disease, Crohn's

disease, allograft rejection, Behcet's disease, sepsis, septic or endotoxin shock and

ischemia.

14. A method of preventing and/or treating a disease associated with overexpression

and/or over-release of IL-17A, comprising administering the drug according to claim

13 to a subject in need thereof.